Membrane penetrating peptides and uses thereof

ABSTRACT

The present invention is directed to membrane penetrating peptides useful as in viv, ex vivo and in vitro intracellular delivery devices for compound of interest. More particularly, the invention involves identification of membrane penetrating peptides which may be used as protein carriers for delivery of a compound of interest to cells, to methods of delivering a compound of interest attached to membrane penetrating peptides to cells.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/227,647, filed Aug. 25, 2001 and GB Application0103110.3, filed Feb. 7, 2001.

FIELD OF THE INVENTION

[0002] The invention relates to membrane penetrating peptides useful asin vitro, ex vivo and in vivo delivery devices for intracellulardelivery of a compound of interest to cells in vitro, ex vivo and invivo, compositions comprising the same and methods of using the same.The invention also includes identification of additional membranepenetrating peptides useful as delivery devices for intracellulardelivery of a compound of interest to cells in vitro, ex vivo and invivo.

BACKGROUND OF THE INVENTION

[0003] The delivery of small molecules, oligonucleotides, and proteinsthrough biological membranes is a major challenge facing therapy andvalidation paradigms. It has recently been established that transducingpeptides derived from Antennapedia, TAT-HIV, and VP22 can penetratebiological membranes, act as cargo vehicles, and target to specificsubcellular compartments. Here we show the identification of a nuclearlocalization sequence (NLS) within human Period 1 (hPER1) circadianprotein that functions as a transducing peptide. More importantly, usingdatabase mining, we have uncovered additional transducing peptidesembedded within the NLS's of other proteins and extend the number ofgene-encoded transducing peptides from 3 to 14. Our data suggest thattransducing peptides are found within NLS's and are prevalent, diverse,and distributed widely throughout the genome. It is well establishedthat certain extracellular and intracellular proteins are targeted tospecific organelles within a cell, transmembrane or secreted from thecell. The biological mechanisms by which intracellular protein targetingoccurs continues to be characterized, but is well recognized that onemechanism for localization occurs by virtue of specific leader sequencecontained within the protein of interest, or intraprotein sequence.Localization of proteins within selected cellular organelles is aided byspecific targeting sequences. A number of nuclear localization sequences(NLSs) have been identified in proteins that permit the protein to betransported or otherwise pass from the cytoplasm into the nuclearmembrane.

[0004] Fusion proteins containing the targeting sequence and another,otherwise non-targeted protein, are localized in the selected cellularorganelle depending on the targeting sequence selected. For example,Ferullo, J. M. and Paget, E. FR 279695, disclose selectivecompartmentalization of an hydroxyphenylpyruvate dioxygenase (HPPD)fused to a signal sequence directing the enzyme to a cellularcompartment other than the cytosol, e.g., a vacuole. Similarly, WO0147950 (Wehrle-Haller, Bernhard M.; Inhof, Beat A) identify a newdeterminant responsible for basolateral targeting and prolonged exposureof cell-surface-anchored growth factors at cell surfaces. The signal isa mono-leucine dependent basolateral sorting signal consisting of theamino acid sequence X1h2X3h4Lp5p6, wherein: X1 represents a polar aminoacid residue or alanine, h2 represents any hydrophobic amino acidresidue, X3 represents any amino acid residue, h4 represents anyhydrophobic amino acid residue, except leucine and isoleucine, Lrepresents a leucine residue, p5 represents any polar amino acidresidue, and p6 represents any polar amino acid. Richardson, A. E., etal., Plant J. (2001), 25(6), 641-649 describe manipulation of the enzymeaspergillus phytase to include the signal peptide sequence from thecarrot extensin gene. The resulting fusion protein was only effectivewhen secreted as an extracellular enzyme into the adjacent soil, andresulted in a 20-fold increase in total root phytase activity intransgenic lines and subsequent improved phosphorus nutrition, such thatthe growth and phosphorus content of the plants was equivalent tocontrol plants supplied with inorganic phosphate. WO 0132894 (Lok, S.)disclose use of the signal anchor domain sequences of type II cellsurface proteins to anchor recombinant proteins into surface oftransfected cells. A characteristic feature of type II cell surfaceproteins is that they are held within the cellular membrane by a singlehydrophobic transmembrane domain and are oriented with their C-terminusoutside the cell.

[0005] More recently, a few proteins have been identified which arecapable of passing through the cellular membrane without requiringactive transport mechanisms or ‘pores’. It is recently established thatmembrane penetrating peptides (MPPs, also known as protein transductiondomain, “PTD”) derived from Antennapedia, TAT, and VP22 can penetratebiological membranes and target to specific subcellular compartments.None of these previously disclosed proteins are derived from mammalianproteins. The present invention is directed to the discovery thatpolypeptides derived from mammalian or yeast proteins nuclearlocalization sequences (NLSs) or overlapping with NLS's are capable ofacting as MPPs, and identification of a specific polypeptide sequencescapable of penetrating cellular membranes, even when conjugated to largeproteins, such as biologically active proteins, or other organiccompounds.

[0006] Nuclear transport is essential to a number of biologicalprocesses including gene expression and cell division, as well as toviral replication, tumorigenesis and tumor cell proliferation. Themechanism of nuclear transport has only recently been characterized indetail and has been shown to involve a number of discrete steps.Proteins that are destined to be transported into the nucleus containwithin their amino acid sequence a short stretch of amino acids termed anuclear localization sequence (“NLS”). These sequences may occuranywhere within the amino acid sequence and are typically four to abouteight amino acids. These sequences are generally basic (i.e., positivelycharged) in nature, however, there has been no consensus sequenceidentified. Thus, there is a wide variety of these sequences that appearto be specific for particular proteins.

[0007] Within the cell, these NLSs may be either masked or unmasked byaccessory proteins or by conformational changes within theNLS-containing protein. An NLS may be masked because it is buried in thecore of the protein and not exposed on the surface of the protein.Unmasking of NLSs, and nuclear translocation of cytoplasmic proteins maybe triggered by phosphorylation, dephosphorylation, proteolyticdigestion, subunit association or dissociation of an inhibitory subunit,or the like. Accordingly, the masking and unmasking of NLSs provides amechanism by which the transport of these cytoplasmic proteins into thenucleus may be regulated. For example, the transcription factor NF-ATcontains nuclear localization sequences which allow NF-AT to translocateto the nucleus in the presence of intracellular calcium, but which areshielded by forming intramolecular associations with other domains inthe NF-AT polypeptide in the absence of calcium.

[0008] Lee, H. C. and Bernstein, H. D. Proc. Natl. Acad. Sci. U.S.A.(2001), 98(6), 3471-3476 studied the mechanism involved for presecretoryproteins such as maltose binding protein (MBP) and outer membraneprotein A (OmpA) that are targeted to the E. coli inner membrane by themolecular chaperone SecB, in contrast to the targeting of integralmembrane proteins by the signal recognition particle (SRP). The authorsfound that replacement of the MBP or OmpA signal peptide with the firsttransmembrane segment of AcrB abolished the dependence on SecB fortransport and rerouted both proteins into the SRP targeting pathway.

[0009] Some proteins contain cytoplasmic localization sequences (CLS),or nuclear export sequences, which ensure the protein remainspredominantly in the cytoplasm. For example, Hamilton, M. H. et al., J.Biol. Chem. (2001), 276(28), 26324-26331 demonstrate that theubiquitin-protein ligase (E3), hRPF1/Nedd4, a component of theubiquitin-proteasome pathway responsible for substrate recognition andspecificity, is capable of entering the nucleus, but the presence of afunctional Rev-like nuclear export sequence in hRPF1/Nedd4 ensures apredominant cytoplasmic localization. The cytoplasmic domains of manymembrane proteins contain sorting signals that mediate their endocytosisfrom the plasma membrane.

[0010] Heineman, T. C. and Hall, S. L. Virology (2001), 285(1), 42-49studied three consensus internalization motifs within the cytoplasmicdomain of VZV gB and determined that internalization of VZV gB, and itssubsequent localization to the Golgi, is mediated by two tyrosine-basedsequence motifs in its cytoplasmic domain. In mammalian cells andyeasts, amino acid motifs in the cytoplasmic tails of transmembraneproteins play a prominent role in protein targeting in the earlysecretory pathway by mediating localization to or rapid export from theendoplasmic reticulum (ER). Hoppe, H. C. and Joiner, K. A. Cell.Microbiol. (2000), 2(6), 569-578.

[0011] The mammalian endopeptidase, furin, is predominantly localized tothe trans-Golgi network (TGN) at steady state. The localization of furinto this compartment seems to be the result of a dynamic process in whichthe protein undergoes cycling between the TGN and the plasma membrane.Both TGN localization and internalization from the plasma membrane aremediated by targeting information contained within the cytoplasmicdomain of furin. Voorhees, P., et al., EMBO J. (1995), 14(20), 4961-75report that there are at least two cytoplasmic determinants thatcontribute to the steady-state localization and trafficking of furin.The first determinant corresponds to a canonical tyrosine-based motif,YKGL (residues 758-761), that functions mainly as an internalizationsignal. The second determinant consists of a strongly hydrophilicsequence (residues 766-783) that contains a large cluster of acidicresidues (E and D) and is devoid of any tyrosine-based ordi-leucine-based motifs. This second determinant is capable ofconferring localization to the TGN as well as mediating internalizationfrom the plasma membrane.

[0012] The trans-Golgi network (TGN) plays a central role in proteinsorting/targeting and the sequence SXYQRL can by itself confersignificant TGN localization. Wong, S. H., and Hong, W. J. Biol. Chem.(1993), 268(30), 22853-62 report detailed mutagenesis of the 32-residuesequence of TGN38, an integral membrane protein confined mainly to theTGN, and determined that the Ser, Tyr, and Leu residues at positions 23,25, and 28, respectively, are essential for TGN localization. When thecytoplasmic 32-residue sequence of TGN38 was fused to the ecto- andtransmembrane domains of glycophorin A (a surface protein), theresulting chimeric protein was localized to the TGN.

[0013] It is well recognized that certain proteins are either onlyactive in a specific organelle, or are capable of different functionsdepending on their localization. For example, appropriate subcellularlocalization is crucial for regulation of NF-κB function. Huang, T. T.,et al., Proc. Natl. Acad. Sci. U.S.A. (2000), 97(3), 1014-1019, showthat latent NF-κB complexes can enter and exit the nucleus inpreinduction states and identified a previously uncharacterized nuclearexport sequence in residues 45-54 of IκBα that was required forcytoplasmic localization of inactive complexes. It appears thatNF-κB/IκBα complexes shuttle between the cytoplasm and nucleus by anuclear localization signal-dependent nuclear import and aCRM1-dependent nuclear export and that the dominant nuclear export overnuclear import contributes to the largely cytoplasmic localization ofthe inactive complexes to achieve efficient NF-κB activation byextracellular signals.

[0014] Nuclear import of classical nuclear localizationsequence-containing proteins involves the assembly of an import complexat the cytoplasmic face of the nuclear pore complex (NPC) followed bymovement of this complex through the NPC and release of the importsubstrate into the nuclear interior. In combination with Ran, two othersoluble factors are thought to be absolutely required to mediate thenuclear import of a protein containing a classical or basic NLS into thenucleus. The first is karyopherin/importin α (Kap α), which binds aclassical NLS and then forms a complex with karyopherin/importin β1(Kapβ1). Adam, S. A., and Gerace, L. (1991) Cell 66, 837-847; Görlich,D., et al. (1994) Cell 79, 767-778; Moroianu, J., et al. (1995) Proc.Natl. Acad. Sci. U.S.A. 92, 2008-2011; Radu, A., et al. (1995) Proc.Natl. Acad. Sci. U.S.A. 92, 1769-1773; Görlich, D., et al. (1995) Curr.Biol. 5, 383-392; Chi, N. C., et al. (1995) J. Cell Biol. 130, 265-274.Kap β1 interacts with nuclear pore complex (NPC) proteins and appears tomediate movement of the import complex through the NPC via theseinteractions. Rexach, M., and Blobel, G. (1995) Cell 83, 683-692; Radu,A., Blobel, G., and Moore, M. S. (1995) Proc. Natl. Acad. Sci. U.S.A.92, 1769-1773; Iovine, M. K., Watkins, J. L., and Wente, S. R. (1995) J.Cell Biol. 131, 1699-1713; Radu, A., Moore, M. S., and Blobel, G. (1995)Cell 81, 215-222. Another protein, p10/NTF2, has also been implicated innuclear import, but its function may only be to take Ran into thenucleus, where it is subsequently needed to disassemble an incomingimport complex. Moore, M. S., and Blobel, G. (1994) Proc. Natl. Acad.Sci. U.S.A. 91, 10212-10216; Paschal, B. M., and Gerace, L. (1995) J.Cell Biol. 129, 925-937; Ribbeck, K., Lipowsky, G., Kent, H. M.,Stewart, M., and Görlich, D. (1998) EMBO J. 17, 6587-6598; Smith, A.,Brownawell, A., and Macara, I. G. (1998) Curr. Biol. 8, 1403-1406.

[0015] Although there is only one Kap α homologue in yeast (SRP1 orKap60), vertebrate cells contain a number of proteins that can bind aclassical NLS and share sequence homology (see Ref. Nachury, M. V.,Ryder, U. W., Lamond, A. I., and Weis, K. (1998) Proc. Natl. Acad. Sci.U.S.A. 95, 582-587, and references therein). These proteins have beengiven a variety of names but can be grouped into three major families.The Kap α1 family contains the human protein NPI-1/importinα1/karyopherin α1/Rch2/hSRP1 and a second related protein importin α6,in addition to the mouse S2 protein. Moroianu, J., et al., (1995) Proc.Natl. Acad. Sci. U.S.A. 92, 2008-2011; Cortes, P., et al., (1994) Proc.Natl. Acad. Sci. U.S.A. 91, 7633-7637; O'Neill, R. E., et al., (1995) J.Biol. Chem. 270, 22701-22704; Kohler, M., et al., (1997) FEBS Lett. 417,104-108; Tsuji, L., et al., (1997) FEBS Lett. 416, 30-34. The secondfamily, Kapα2, contains human Rch1/hSRP1/importin α2/karyopherin α2 andthe mouse protein pendulin/PTAC 58. Görlich, D., Prehn, S., Laskey, R.A., and Hartmann, E. (1994) Cell 79, 767-778; Cuomo, C. A., Kirch, S.A., Gyuris, J., Brent, R., and Oettinger, M. A. (1994) Proc. Natl. Acad.Sci. U.S.A. 91, 6156-6160; Kussel, P., and Frasch, M. (1995) Mol. Gen.Genet. 248, 351-363; Imamoto, N., Shimamoto, T., Takao, T., Tachibana,T., Kose, S., Matsubae, M., Sekimoto, T., Shimonishi, Y., and Yoneda, Y.(1995) EMBO J. 14, 3617-3626;, K., Mattaj, I. W., and Lamond, A. I.(1995) Science 268, 1049-53. The third family, Kapα3, consists of thetwo human proteins, QIP-1/importin α3 and KPNA3/hSPR1 γ/hSRP4, and themouse proteins Q1 and Q2. Nachury, M. V., et al., (1998) Proc. Natl.Acad. Sci. U.S.A. 95, 582-587; Kohler, M., et al., (1997) FEBS Lett.417, 104-108; Tsuji, L., et al., (1997) FEBS Lett. 416, 30-34; Takeda,S., et al., (1997) Cytogenet. Cell Genet. 76, 87-93; Seki, T., et al.,(1997) Biochem. Biophys. Res. Commun. 234, 48-53; Miyamoto, Y., et al.,(1997) J. Biol. Chem. 272, 26375-26381. Each of these classes shareabout 50% homology with each other and to the yeast SRP1, and each ofthese mammalian proteins has been shown to be capable of mediating theimport of one or more classical NLS-containing proteins. Nachury, M. V.,et al., (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 582-587; Sekimoto, T.,et al., (1997) EMBO J. 16, 7067-7077; Nadler, S. G., et al., (1997) J.Biol. Chem. 272, 4310-4315; Prieve, M. G., et al., (1998) Mol. Cell.Biol. 18, 4819-4832.

[0016] Stat-1 import is mediated by Kapα1/NPI-1 but not Kapα2/Rch1, butactivated Stat-1 appears to bind to a COOH-terminal region of Kapα1distinct from the NLS binding Armadillo repeats. The binding differencesof the different Kapαs to RCC1 observed appear to be due solely to theNLS on RCC1 and therefore probably due to the NLS binding region ofKapα3. Sekimoto, T., et al., (1997) EMBO J. 16, 7067-7077. Kamei, Y., etal., (1999) J. Histochem. Cytochem. 47, 363-372 showed that, in mice,the Kapα3 homologue is expressed in many tissues and theorized thatKapα3 may play a role in importing “a limited number of uniquekaryophilic proteins, such as helicase Q1.” The results provided byTalcott, B. and Moore, M. S., 2000 J Biol Chem, 275(14) 10099-10104suggest that RCC1 should be included in the group of proteins that useKapα3 to mediate their nuclear import.

[0017] U.S. Pat. No. 6,191,269 teaches the existence of a nuclearlocalization sequence contained within the cDNA sequence of theN-terminal IL-1 alpha propiece, T76-NGKVLKKRRL, which hadcharacteristics of a nuclear localization sequence (NLS) and couldmediate nuclear localization of the propiece (Stevenson et al. (1997)Proc. Natl. Acad. Sci. USA 94:508-13). Introduction of the cDNA encodingthe N-terminal IL-.alpha. propiece into cultured mesangial cellsresulted in nuclear accumulation (Stevenson et al. id).

[0018] U.S. Pat. No. 5,877,282 teaches that the antennapedia homeodomainsignal sequence peptide is the amino acid sequence RQIKIWFQNRRMKWKK; thefibroblast growth factor signal sequence peptide is AAVALLPAVLLALLA; theHIV Tat signal sequence peptide is the amino acid sequenceCFITKALGISYGRKKRRQRRRPPQGSQTH.

[0019] Schwartze, S. R., et al., Science 285:1569-1572 (1999) reportdelivery of an ip injected reporter protein, 116 kD beta-galactosidase,as a TAT fusion protein into tissues and across the blood-brain barrier.Schwartze used an 11 amino acid protein transduction domain (PTD)derived from HIV tat protein with an N-terminal fluoresceinisothiocyanate (FITC)-Gly-Gly-Gly-Gly motif. The authors report thatearlier attempts to transduce beta-Gal chemically cross-linked to theTAT PTD resulted in sporadic and weak beta-Gal activity in a limitednumber of tissues. They speculate that the improved transduction was dueto the in-frame fusion and purification strategy used.

[0020] Nuclear localization of IFNγ is mediated by a polybasic NLS inits C terminus, which is required for the full expression of biologicalactivity of IFNγ, both extracellularly and intracellularly. Subramaniam,Prem S., et al., J. Cell Sci. (2000), 113(15), 2771-2781. This NLS isthought to play an integral intracellular role in the nucleartranslocation of the transcription factor STAT1α activated by IFNγbecause treatment of IFNγ with antibodies to the C-terminal region(95-133) containing the NLS blocked the induction of STAT1α nucleartranslocation, but these antibodies had no effect on nucleartranslocation of STAT1α in IFNα treated cells. A deletion mutant ofhuman IFNγ, IFNγ(1-123), which is devoid of the C-terminal NLS regionwas biologically inactive, but was still able to bind to the IFNγreceptor complex on cells with a K_(d) similar to that of the wild-typeprotein. Deletion of the NLS specifically abolished the ability ofIFNγ(1-123) to initiate the nuclear translocation of STAT1α, which isrequired for the biological activities of IFNγ following binding to theIFNγ receptor complex. A C-terminal peptide of murine IFNγ,IFNγ(95-133), that contains the NLS motif, induced nuclear translocationof STAT1α when taken up intracellularly by a murine macrophage cellline. Deletion of the NLS motif specifically abrogated the ability ofthis intracellular peptide to cause STAT1α nuclear translocation. Incells activated with IFNγ, IFNγ was found to as part of a complex thatcontained STAT1α and the importin-α analog Npi-1, which mediates STAT1αnuclear import. The tyrosine phosphorylation of STAT1α, the formation ofthe complex IFNγ/Npi-1/STAT1α complex and the subsequent nucleartranslocation of STAT1α were all dependent on the presence of the IFNγNLS.

[0021] The peptide representing amino acids 95-132 of IFN-γ(IFN-γ(95-132)), containing the polybasic sequence ¹²⁶RKRKRSR¹³², wascapable of specifying nuclear uptake of the autofluorescent protein,APC, in an energy-dependent fashion that required both ATP and GTP.Nuclear import was abolished when the above polybasic sequence wasdeleted. Subramaniam, P., et al., 1999 J Biol Chem 274(1) 403-407. Apeptide containing the prototypical polybasic NLS sequence of the SV40large T-antigen was also able to inhibit the nuclear import mediated byIFN-γ(95-132), suggesting that the NLS in IFN-γ may function through thecomponents of the Ran/importin pathway utilized by the SV40 T-NLS.Intact IFN-γ, when coupled to APC, was also able to mediate its nuclearimport, and this nuclear import was blocked by the peptide IFN-γ(95-132) and the SV40 T-NLS peptide, suggesting that intact IFN-γ wasalso transported into the nucleus through the Ran/importin pathway.

[0022] Nuclear proteins are imported into the nucleus through aqueouschannels that span the nuclear envelope called nuclear pore complexes(NPCs). Although ions and molecules less than ˜20-40 Da can diffusepassively through the nuclear pore complexes, larger proteins aretransported by saturable pathways that are energy- and signal-dependent.The signals that specify nuclear protein import (NLSs)1 are commonlyshort stretches of amino acids rich in basic amino acid residues,although other classes of NLSs have been described recently. The initialstep in the import of proteins containing basic amino acid-type NLSsoccurs in the cytosol, where the NLS-containing proteins are bound to areceptor (variously called the NLS receptor, importin α, and karyopherin(13). The substrate-receptor complex then associates with thecytoplasmic face of the nuclear pore complexes, and with theparticipation of other cytosolic factors, is transported through a gatedchannel in the nuclear pore complexes to the nuclear interior. The invivo events of NLS-mediated nuclear import can be duplicated in an invitro system using digitonin-permeabilized cells supplemented withcytosolic extracts and ATP (14). Transport in this in vitro assay isblocked by the same inhibitors that block in vivo import, is rapid, andis easily quantified.

[0023] The NLS the sequence NYKKPKL in the N-terminus of fibroblastgrowth factor (FGF)-1, the precursor for acidic FGF, has been proposedto affect the long term activities of FGF-1 through its function as anuclear translocation signal or its role in stabilization of thestructure required to sustain binding and activation of thetransmembrane receptor kinase. Luo, Y., et al., J. Biol. Chem. (1996),271(43), 26876-26883. For example, concurrent with a marked increase independence on exogenous heparin for optimal activity, sequentialdeletion of residues in the NYKKPKL sequence in FGF-1 resulted in aprogressive loss of thermal stability, resistance to protease, mitogenicactivity, and affinity for the transmembrane receptor. The largestchange resulted from deletion of the entire sequence through thelysine-leucine residues. In the presence of sufficiently highconcentrations of heparin, the deletion mutants exhibited mitogenicactivity equal to wild-type FGF-1.

[0024] Although FGF-1 contains an NTS, nuclear translocation requires anexogenous and not an endogenous pathway. The NTS of FGF-1, NYKKPKL, isable to direct the expression of the bacterial β-galactosidase (βgal)gene to the nucleus of transfected NIH 3T3 cells, but this NTS is unableto target either FGF-1 itself of a FGF-1-βgal fusion protein into thenucleus, suggesting that FGF-1 may contain an additional sequence whichprevents endogenously expressed FGF-1 from being translocated into thenucleus. Zhan, X., et al., Biochem. Biophys. Res. Commun. (1992),188(3), 982-91.

[0025] Interferon-γ (IFN-γ), a protein that uses the Jak-Stat pathwayfor signal transduction, translocates rapidly to the nucleus in cellstreated extracellularly with the cytokine. An NLS has been identifiedand characterized in the C-terminus of human and murine IFN-γ. Larkin,J., et al., J. Interferon Cytokine Res. (2001), 21(6), 341-348 reportthat human IFN-γ (HuIFN-γ) contains a second NLS at an upstream site.The primary sequence, analogous with the NLS sequence identified inmurine IFN-γ, representing amino acids 122-132 of HuIFN-γ was capable ofmediating the nuclear import of the autofluorescent proteinallophycocyanin (APC) in an energy-dependent manner. The secondsequence, representing amino acids 78-92 of HuIFN-γ, was also capable ofmediating the nuclear import of APC in an energy-dependent manner but toa greatly reduced extent. The nuclear import of both sequencesconjugated to APC was strongly blocked by competition with unconjugatedHuIFN-γ(122-132). Competition by the sequence HuIFN-γ(78-92) effectivelyblocked the import of APC-conjugated HuIFN-γ(78-92) but, at the sameconcentration, was not capable of inhibiting the nuclear import ofAPC-conjugated HuIFN-γ(122-132), suggesting that HuIFN-γ(78-92) was aless efficient NLS than HuIFN-γ(122-132). This is consistent with >90%loss of antiviral activity of HuIFN-γ lacking the downstream NLS in122-132. The nuclear import of APC-conjugated HuIFN-γ(122-132) wasinhibited by a peptide containing the prototypical polybasic NLS of theSV40 T NLS, which suggests that the same Ran/importin cellular machineryis used in both cases.

[0026] There appears to be strong conservation of the NLS motif as amechanism for nuclear localization. Evolution seemed to have used partof the existing DNA-binding mechanism when compartmentalizingDNA-binding proteins into the nucleus. Cokol, M., et al., EMBO Rep.(2000), 1(5), 411-415 estimate that greater than 17% of all eukaryoticproteins may be imported into the nucleus, and after analyzing a set of91 experimentally verified NLSs from the literature and expanding thisset to 214 potential NLSs through iterated “in silico mutagenesis”. Thisfinal set matched in 43% of all known nuclear proteins and in no knownnon-nuclear protein. Cokel et al found an overlap between the NLS andDNA-binding region for 90% of the proteins for which both the NLS andDNA-binding regions were known, but only 56 of the 214 NLS motifsoverlapped with DNA-binding regions. These 56 NLSs enabled a de novoprediction of partial DNA-binding regions for approximately 800 proteinsin human, fly, worm and yeast.

[0027] More recently, it has been reported that NLS signal peptide caninduce structural changes of DNA. The plant enzyme, glutaminyl-tRNAsynthetase (GlnRS) from Lupinus luteus, contains an NLS at theN-terminal, a lysine rich polypeptide, KPKKKKEK. Krzyzaniak, A., et al.,Mol. Biol. Rep. (2000), 27(1), 51-54. Two synthetic peptides (20 and 8amino acids long), derived from the NLS sequence of lupin GlnRS interactwith DNA. In addition, the shorter 8 amino acid peptide caused the DNAto change its conformation from the B to the Z form. This observationclearly suggests that the presence of the NLS polypeptide in a leadersequence of GlnRS is required not only for protein transport intonucleus but also for regulation of a gene expression. This is the firstreport suggesting a role of the NLS signal peptide in structural changesof DNA.

[0028] Typically there is strong conservation of the NLS sequence withinspecies. For example, the NLS in the N-terminal region of Smad 3protein, the major Smad protein involved in TGF-β signal transduction,has a basic motif Lys⁴⁰- Lys-Leu-Lys-Lys⁴⁴, which is conserved among allthe pathway-specific Smad proteins, and is required for Smad 3 nuclearimport in response to ligand. Smad proteins are intracellular mediatorsof transforming growth factorβ (TGF-β) and related cytokines. Xiao, Z.,et al., J. Biol. Chem. (2000), 275(31), 23425-23428 identified the rolethe NLS plays in nuclear localization. The authors demonstrated that theisolated Smad 3 MH1 domain displays significant specific binding toimportin β, which is diminished or eliminated by mutations in the NLS.Full-size Smad 3 exhibits weak but specific binding to importin β, whichis enhanced after phosphorylation by the type I TGF-β receptor. Incontrast, no interaction was observed between importin α and Smad 3 orits MH1 domain, indicating that nuclear translocation of Smad proteinsmay occur through direct binding to importin β. The authors concludethat activation of all of the pathway-specific Smad proteins (Smads 1,2, 3, 5, 8, and 9) exposes the conserved NLS motif, which then bindsdirectly to importin β and triggers nuclear translocation.

[0029] In all cells, the lipid bilayer of cell membranes serves as aselective barrier for the passage of charged molecules, with theinternalization of hydrophilic macromolecules being achieved throughclassical transport pathways (Hawiger, J., Curr Opin Chem Biol. 3, 89-94(1999), Schwartze, S. R., et al., Trends in Cell Biology 10, 290-295(2000)). These classical mechanisms of internalization involvereceptor-mediated endocytosis or transporter dependent uptake (Cleves,A. E., Current Biology 7, R318-R320 (1997)). In contrast, an increasingnumber of molecules have been discovered that lack classical importand/or export signals (Cleves, A. E., Current Biology 7, R318-R320(1997)). These molecules gain direct access to either cytoplasmic ornuclear compartments using unconventional processes of which themechanisms remain largely unknown. These novel mechanisms are generallytermed “nonclassical” and refer to transport pathways being used thatare atypical. Relevant examples of this latter type are found in thegene-encoded proteins of HIV-1 TAT (Frankel, A. D. and Pabo, C. O. Cell55,1189-1193 (1988)), herpes virus VP22 (Elliott, G. and O'Hare, P. Cell88, 223-233 (1997)), and Antennapedia, Antp (Derossi, D., et al., J.Biol. Chem. 269,10444-10450 (1994)). It is now well established that thefull-length proteins of HIV-1 TAT (Helland D. E., et al., J Virol 65,4547-4549 (1991)), and VP22 (Pomeranz L. E. and Blaho J. A., J Virol 73,6769-6781 (1999)) rapidly translocate into and out of cellularmembranes. In fact, distinct peptide regions have been identified withinboth of these proteins that are capable of translocating into cellularcompartments either alone or in combination with chimeric cargopeptides, and proteins (Lindgren, M., et al., Trends Pharmacol Sci. 3,99-103 (2000), Derossi, D., et al, Trends Cell Biol., 8, 84-87 (1998),Prochiantz A., Current Opinion in Cell Biology 12, 400-406 (2000),Steven R. Schwarze, S. R., et al., Trends in Cell Biology 10, 290-295(2000)). In contrast, full-length Antp protein has not been shown totraverse biological membranes; however, a 16 amino acid syntheticpeptide derived from within its coding region does possess potentmembrane penetrating abilities (Derossi, D., et al, Trends Cell Biol.,8, 84-87 (1998)). The accepted view of atypical transport used by thesemolecules has been termed “transduction” (Schwarze, S. R., et al.,Trends in Cell Biology 10, 290-295 (2000)), and is currently defined asan extremely rapid membrane transport pathway that is receptor andenergy independent, and can occur at 4 C. in all cell types (Schwarze,S. R. and Dowdy, S. F. Trends Pharmacol. Sci. 21, 45-48 (2000)).Interestingly, these three proteins are all nuclear proteins involved intranscriptional regulation, and their respective transducing peptidesconsist of strings of amino acids rich in arginine and lysine (Lindgren,M., et al., Trends Pharmacol Sci. 3, 99-103 (2000), Schwarze, S. R. andDowdy, S. F. Trends Pharmacol. Sci. 21, 45-48 (2000)). However,irrespective of these similarities, these transducing peptides possessmany different characteristics such as amino acid sequence, length ofthe sequence, cellular localization, and potency of membranepenetration. Thus, though each transducing sequence can penetrate cellsand tissues, it has not been established whether they use the identicalatypical transport mechanisms.

[0030] Finally, U.S. Pat. No. 6,022,950 teaches the use of a hybridmolecule of a portion of the binding domain of a cell-bindingpolypeptide ligand effective to cause said hybrid protein to bind to acell of an animal, a translocation domain of naturally occurring proteinwhich translocates said third part across the cytoplasmic membrane intothe cytosol of the cell; and a chemical entity to be introduced into thecell. However, the patent teaches translocation domains of toxins.Naturally-occurring proteins which are known to have a translocationdomain include diphtheria toxin and Pseudomonas exotoxin A, and mayinclude other toxins and non-toxin molecules, as well. The translocationdomains of diphtheria toxin and Pseudomonas exotoxin A are wellcharacterized (see, e.g., Hoch et al., Proc. Natl. Acad. Sci. USA82:1692-1696, 1985; Colombatti et al., J. Biol. Chem. 261:3030-3035,1986; and Deleers et al., FEBS 160:82-86, 1983), and the existence andlocation of such a domain in other molecules may be determined bymethods such as those employed by Hwang et al., Cell 48:129-136, 1987;and Gray et al., Proc. Natl. Acad. Sci. USA 81:2645-2649, 1984.

[0031] Given the considerable body of literature teaching controlmechanisms of cellular localization, the proteins involved in regulationof intracellular transport, the different properties and controlmechanisms for plasma membrane and the nuclear envelope, it isunexpected that polypeptides derived from mammalian proteins couldtransduce through the plasma membrane using nonclassical mechanisms andthus could be useful as membrane penetrating peptides useful as invitro, ex vivo and in vivo delivery devices of a compound of interest.There is also considerable literature teaching non-protein derivedmethods for delivering a compound of interest into cells, for exampleelectroporation, membrane fusion with liposomes, high velocitybombardment with DNA-coated microprojectiles, incubation withcalcium-phosphate-DNA precipitate, DEAE-dextran mediated transfection,infection with modified viral nucleic acids, and direct microinjectioninto single cells, usually ova and the like. Each of these methods isrelatively inefficient, resulting in relatively low percentage of thecells containing the delivered compound of interest and most of themethods are clearly not capable of realistic in vivo delivery. Many ofthe methods are toxic to the cells, resulting in relatively highapoptosis. Therefore, there is a considerable need for simple and moreefficient delivery of compounds of interest into cells.

SUMMARY OF THE INVENTION

[0032] The present invention is directed to polypeptides derived frommammalian and yeast proteins useful as a carrier for in vitro, ex vivoand in vivo delivery a compound of interest. The invention also providescompositions containing the same, and methods of delivering a compoundof interest in vitro, ex vivo and in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1.(A). Schematic diagram of hPER1 fusion constructs showingthe locations of the PAS, cytoplasmic localization, and nuclearlocalization sequence (NLS, but indicated as nuclear localization domain(NLD) in Figure). The name and the position of the fusion constructs arelisted on the left. The number indicates the first and last amino acidresidues in the hPER1 protein. The principal sites of accumulation ofeach fusion protein are summarized on the right, (n) nuclear, (no)nucleoli, (c) cytoplasmic, (diff) diffuse. All constructs wereN-terminally tagged with EYFP. The alignment human and mouse PER1-NLS isshown at the bottom.

[0034]FIG. 1.(B). Cellular localization of hPER1 fusion proteins asdescribed in FIG. 1A, above, in living cells. CHO cells were transienttransfected with the fusion constructs indicated on the top of eachpanel and the subcellular localization of EYFP reporters (green) wasdirectly visualized using fluorescent microscopy 10 h post-transfection.EYFP vector alone is used as control (see 5. EYFP-VECTOR)

[0035]FIG. 2.(A). Membrane penetration assay in CHO cells. N-terminalbiotinylated synthetic peptides hPER1-PTD, Flag-hPER1-PTD, Flag-TAT-PTD(positive control), and Flag-Flag (negative control) were assayed fortheir ability to penetrate cellular membranes in living CHO cells inculture. The subcellular localization of internalized peptides wasdetermined using a two color staining method, either Streptavidin-Alexa594 (red) or anti-flag mAb (green). The third column is an overlay(yellow). Confocal microscopy was employed to further confirmintracellular and intranuclear localization. Single section of confocalimaging is shown.

[0036]FIG. 2.(B). Nuclear targeting of biotinylated peptides hPER1-NLD(also known as hPER1-PTD) compared with TAT-PTD and Flag-Flag (negativecontrol) using Streptavidin Alexa-594 fluorescence (green). Hoechst33258 at 5 ng/ml was used to stain the nucleus (blue, middle column).The third column is an overlay of confocal imaging.

[0037]FIG. 3. Alanine scanning of hPER1-PDTs. Biotinylated hPER1-NPDswere synthesized with a single amino acid residue substitution at theindicated position with an alanine and assayed for membrane penetrationin CHO cells. Cells were incubated for 10 minutes at 37 C. at a peptideconcentration of 10 μM followed by washing, fixation, permeablization,and then detected with labeled Streptavidin Alexa-594 (red, 2 μg/ml) for15 minutes at the RT. Control peptide was from hPER1 N-terminal aminoacids residues 486-500.

[0038]FIG. 4. Activation of serotonin 5HT2A receptor with hPER1-MPPfusion peptide. (A). hPER1-MPP and TAT-PTD peptides were synthesizedalone or in fusion with either the first intracellular loop I1(SLEKKLQNATN), or the C-terminal Transmembrane 7 domain, TM7(KTYRSAFSRYIQYKENKKPLQLI) derived from the 5HT2A receptor, genebankaccession numbr, M86841). Receptor activities was assayed using standardFLIPR analysis and measuring endogenous and exogenous Ca⁺² levels.Peptide designations are as follows: T (TAT-PTD), P (hPER1-MPP), I1(intracellular loop 1), T-I1 (TAT-PTD-I1), P-I1 (hPER1-MPP-I1), TM7(C-terminal domain), TTM7 (TAT-PTD-TM7), PTM7 (hPER1-MPP-TM7), and S(Serotonin).

[0039]FIG. 4(B). Dose response of PTM7 (closed circles) and TTM7 (closeddiamonds) peptides. Serotonin (control, open triangle) was used at themaximum receptor stimulatory concentration of 10 μM.

[0040]FIG. 5. Identification of additional PTDs. Putative PTD sequenceswere searched using a combined bioinformatics method that includedSwissPro, PRF, PIR-Protein info Resource, PDB with peptides sequencestranslated from the annotated protein coding region in GenBank with“transcription factor” as the key word. We initially searched for allknown or putative NLS's. Secondly, we employed the PHI-BLAST(Pattern-Hit Initiate BLAST) to search for the degenerative patternoccurrence [R/H/K]-[R/H/K]-[R/H/K]-[R/H/K], (X)n where n is an integerof 4 or larger and X each time is independently selected to be eitherarginine, histidine, or lysine. 7374 putative PTD sequences wereidentified. From the two searches we synthesized (A) biotinylatedpeptides to these sequences or (B) created in frame fusion proteins withGFP and transfected CHO cells. 9 of the 12 peptides were found totransduce, and all sequences localize to the nucleus in transfectedcells. hPER1-PTD, hPER3-PTD, and TAT-PTD peptides were used as positivecontrols. Six positive sequences and 2 negative sequences are shown.Numbers represent the amino acid residues within the parental proteinsequence and Gene bank accession numbers for these proteins areindicated as follows: (M24899, human Thyroid hormone alpha-1; L12699,human Homeobox protein Engrailed 1 HME1; X16416, human Proto-oncogenetyrosine protein kinase ABL1;; Q02575, human HEN1/NSLC1; Q02577, humanHEN2/NSLC2; AAA74561, rat HNF-3; CAB65887, Drosophila cAMP dependenttranscription factor). Three negative peptides are (V01512, c-Fos;AAD53184, human cyclin L ania-6a; CAB66914, Arabidopsis β-ziptranscription factor).

[0041]FIG. 6. hPER-PTD cargo's β-Galactosidase into cells: At least onefeature of HIV TAT transducing peptide is its ability to cargo proteinsinto cells and tissues. We therefore sought to determine if hPER1transducing peptide could cargo beta galactosidase into cells. Toperform this experiment, we followed a protocol by Frankel et al. 1989(19):7397-401, whereby, we chemically linked hPER1-PTD or hPER-PTD R7Ato full length β-galactosidase and assayed for the ability of theseconjugates and beta-galactosidase protein alone to transduce into CHOcells. As shown in the FIG. 6, left, cells incubated with hPER-PTDP-galactosidase fusion showed positive enzymatic activity forβ-galactosidase as indicated by the blue color in the cells after theaddition of X-gal. However, neither hPER-MPP R7A β-galactosidase(center) nor β-galactosidase protein (right) alone was able to enter thecells as indicated by a no blue staining reactivity after the additionof X-gal. These data indicate that like TAT peptide, hPER1-PTD can cargoa large (120 kD) protein into cells.

DETAILED DESCRIPTION OF THE INVENTION

[0042] The present invention is based on discovery that human Period1(hPER1) protein contains an NLS which has now also been identified as anMPP and is useful as a delivery device for intracellular delivery of acompound of interest. hPER1 is involved in regulation of the circadianrhythm and the capacity of hPER1 to translocate to adjacent cells may becritical to its overall biological function of regulating circadianrhythm. The NLS identified within hPER1 does not fit within previouslyidentified NLS sequences, and its identification has resulted inidentification of an algorithm for searching for other NLS sequenceswhich may also function as MPPs.

[0043] Period 1 (hPER1) is a nuclear protein involved withtranscriptional regulation. It is an essential component in the “gears”of the biological clock (Brown, S. A., and Schibler, U., Current Opinionin Genetics & Development 9, 588-594 (1999), Dunlap, J. C., Cell 96,271-290 (1999)), and studies in mice have shown that nuclear entry ofPER1 is essential for the down regulation of CLOCK/BMAL transcriptionalcomplexes (Gekakis N, et al., Science 280, 1564-1569. (1998), Yagita,K., et al., Genes Dev 14,1353-1363 (2000), Lowrey, P. L., et al.,Science 288, 483-492 (2000)). However, to date, the functional NLS forhuman PER1 has not been elucidated. The present inventors identified theNLS within hPER1, and demonstrate that the 16 amino acid and 13 aminoacid sequence, see FIG. 3. hPER1-NLS peptide, hPER1-MPP, has potentmembrane penetrating ability. This work results in the identification offour additional MPPs also derived from nuclear proteins.

[0044] PER1 is a central component in the circadian clock, and itsnuclear entry plays an important role in the regulation of dailyoscillations (Jin, X., et al., Cell 96, 57-68 (1999), Sangoram, A. M.,et al., Neuron 21, 1101-13 (1998 )). Using deletion and fusion proteinanalysis, we identified a NLS that is necessary and sufficient for hPER1nuclear localization. This functional analysis was necessary because theNLS of hPER1 does not conform to classical nuclear localizing consensusmotifs; and therefore, was not identified using standard NLS searchprocedures. We show that a single copy of hPER1-NLS is sufficient forinducing nuclear localization of a reporter protein and of tagged hPER1fragments (P1-F2 to P1-F7) in transfected cells. The PER1-NLS is locatedbetween amino acids (830-845) of hPER1, is embedded within a string of13 amino acids rich in arginine, histidine, and lysine (see Table 1)that is not found in other PERs or other nuclear proteins in availabledatabases. Therefore, though PERs 2 and 3 are nuclear proteins (Jin, X.,et al., Cell 96, 57-68 (1999)), they apparently use alternativesequences and or mechanisms for their nuclear import.

[0045] Peptide fragments of a limited number of nuclear proteins thatare rich in basic residues have been shown to penetrate into cellularmembranes in a receptorless, energy-independent fashion. Sequences fromthree such proteins, TAT, Antp, and VP22 have been demonstrated topossess the ability to penetrate and cargo fusion molecules into cellsand tissues by an as yet undefined mechanism. See, for example, U.S.Pat. Nos. 5,804,604, 5,747,641, 5,674,980, 5,670,617 and 5,652,122issued to Frankel et al., which teach the use of a nine-amino acid HIVTAT-derived polypeptide (Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg) forintracellular delivery of cargo molecules.

[0046] The similarities between hPER1, the hPER1-NLS, and other MPPsprompted us to investigate whether or not hPER1-MPP could have membranepenetrating capability. The immunohistochemical and cytological datapresented herein indicates that the hPER1-MPP functions as a MPP in avariety of cell types. hPER1-MPP demonstrated intense focal staining inthe nuclear plasma as well as in the nucleolus, suggesting that thesubnuclear address of hPER1-MPP is different from the hPER1 (P1-FL)protein that was diffused in the nucleus but not concentrated in thenucleolus. The cellular penetration of hPER1-MPPs is not blocked evenunder the conditions of reversing the sequence (reversed hPER1-MPP),adding negatively charged residues or pre-fixing cells with 4% PFA,unpublished observation, the latter supports the idea that penetrationis receptor and membrane independent. These results are in contrast toother peptide classes that have been described that are derived fromsignal peptide sequences (Hawiger, J., Curr Opin Immunol. 9, 189-94(1997)), DNA antibodies (Deng, S. X., et al., Int Immunol. 12, 415-423(2000)), and other protein domains (Lindgren, M., et al., TrendsPharmacol Sci. 3, 99-103 (2000)) that bind and cross the cell membranesusing slow, temperature, energy, and receptor dependent mechanisms.

[0047] The identification of other MPPs, has been limited by our lack ofunderstanding the mechanisms and structural requirements necessary formembrane peptide penetration. The likelihood that a specific peptidestructure and/or charge is important for membrane penetration isdemonstrated in the alanine scanning experiments whereby a single aminoacid change at arginine 7 appears to be critical for MPP potential. Bycomparing wild-type hPER-MPP to modified P1-R7A, in live cells orpre-fixed and permeabilized cells (data not show), P1-R7A is onlydefective in penetration but not in nuclear targeting once the cellshave been permeabilized. This finding suggests that arginine 7 has amajor role in structure based penetration, and thus provides a usefulmodel for the future structure-function studies. No structuraldeterminants for TAT peptide have been described, but in the case ofAntp, replacing the two tryptophan residues with two phenylalaninesabolishes penetration (Le Roux, I., et al., Proc Natl Acad Sci USA. 90,9120-9124 (1993)). Since hPER1-MPP does not contain any tryptophanresidues, membrane penetration between these two peptides may occur bydifferent mechanisms.

[0048] Full-length HIV TAT and VP22, both of which lack classicalsecretary signal sequences and are therefore exported by non-classicalmechanisms, can also be imported “by transduction”, into cells in anon-classical manner (Prochiantz A., Current Opinion in Cell Biology 12,400-406 (2000)). Therefore, it is interesting to speculate that perhapshPER1 distributes circadian clock information to adjacent SCN neurons orto circadian output pathways by “transduction” mechanisms similar tofull-length TAT and VP22 proteins. However, simply having membranepenetrating sequences within the body of a protein does not necessarilyconfer membrane penetrating capability, as full-length Antp protein isneither exported from nor imported into cells. Thus, the non-classicalpenetration of the Antp peptides into the cells is unlikely to havephysiological relevance, and like Antp, there is no evidence to suggestthat full-length hPER1 is a cell membrane penetrating protein. However,these findings did encourage us to search for other MPP-containingproteins. By searching protein databases with an algorithm designed toidentify strings of basic residues within nuclear proteins, we uncoveredhundreds of proteins that contained potential membrane penetratingpeptide regions and found 4 additional MPPs from several species (seeFIG. 5). These and additional database mining searches suggest thatMPP-like sequences are common, and present within a wide variety ofproteins. However, like many putative NLSs that do not always confernuclear localization when fused to reporter sequences (Moroianu, J., JCell Biochem. 32-33, 76-83 (1999)), any potential MPPs must befunctionally determined experimentally. Though it seems clear thateither transducing or non-transducing proteins can encode MPP regions,the interesting question that remains is whether or not proteinscontaining MPP-like sequences use these domains to rapidly translocateintracellularly into cellular domains to activate normal physiologicalprocesses. The efficiency associated with the transduction phenomenamight be particularly useful where the rapid delivery of intercellularinformation is critical, as may be the case in cell synchronization,development, and differentiation paradigms.

[0049] The ability for MPPs to cargo molecules to intracellularcompartments is becoming well-established (Lindgren, M., et al., TrendsPharmacol Sci. 3, 99-103 (2000), Derossi, D., et al, Trends Cell Biol.,8, 84-87 (1998)). Similar to other MPPs, hPER1-MPP and other MPPsidentified herein can deliver compounds of interest, such as largemolecules, i.e., peptides and proteins, lipids, polysaccharides, otherorganic molecules, rapidly and efficiently into cells. The datapresented herein demonstrates that hPER1-MPP in fusion with eitherserotonergic and/or adrenergic 7TM-receptor derived peptides mimic theeffects of ligand activated receptors (see FIG. 4, and data not shown),confirming that hPER1-MPP translocates compounds of interest tointracellular compartments, and supports the idea that physiologicallyrelevant signaling can be initiated by MPPs linked to compounds ofinterest. Using the methods described herein, the present invention maybe expanded to provide target validation using MPPs linked to targets,and/or therapeutic strategies using MPPs linked to specific enzymes orreceptors as a method of altering, correcting or compensating fordysfunctional enzyme performance or within pathways. In addition,therapeutic strategies using MPPs linked to specific receptors may beused as a method of altering, correcting or compensating fordysfunctional receptor, low expression of normal or abnormal receptors.

[0050] Taken together, the results provided herein demonstrate an MPPencoded by a mammalian protein and more specifically, a human nuclearprotein, whose cellular penetration is membrane independent and likelydepends on the peptide structure. hPER1-MPP targets to specificsubnuclear sites, but has the potential to efficiently deliver othermacromolecules to intracellular targets.

[0051] More importantly, this invention also provides the first examplefor mapping a novel MPP based on a NLS domain, and suggests that manyMPP-like regions are contained within a wide variety of proteins. Thedata provided herein demonstrate that an MPP may be based on part of anNLS, or overlap with part of the NLS, or alternatively, may be a novelpeptide.

[0052] Methods of identifying NLS sequences are well known in the art,and include NLSs previously identified as conferring the ability of thenative protein to enter the nucleus, or is a putative NLS based onsubstantial sequence homology with a previously identified NLS.Alternatively, the NLS may be identified by sequence deletionexperiments. See for example, Luo J C, Shibuva M A variant of nuclearlocalization signal of bipartite-type is required for the nucleartranslocation of hypoxia inducible factors (1alpha, 2alpha and 3alpha).Oncogene. 2001 Mar 22;20(12):1435-44 or Hodel M R, Corbett A H, Hodel AE. Dissection of a nuclear localization signal. J Biol Chem. 2001 Jan12;276(2):1317-25.

[0053] Preferred membrane penetrating peptides (MPPs, also known aspeptide transduction domain or ‘PTD’) of the present invention are smallpolypeptides, and may be derived from an NLS, or overlapping with anNLS, of a mammalian or yeast protein. Preferred mammalian proteins arethose of human, primate, murine or rat species. It is generallypreferred to use the same species for the NLS-derived protein as thecell to be treated. Human species are especially preferred as theNLS-derived protein when being used to treat human cells. NLSs may befound within a broad class of enzymes, and is not limited to nuclearproteins, transcription factors, cytokines and kinases. Preferred MPPsare those derived from nuclear proteins or transcription factors.Alternatively, MPPs of the present invention are small polypeptidescomprising a sequence —(X—X—X—X)_(n)— where n is an integer 1 to 7, andX each time is independently selected from the group consisting ofarginine, histidine or lysine. It is preferred that small MPPs are used,and therefore, it is preferred that n is an integer 1 to 5, and morepreferred that n is an integer 1 to 3. Selected embodiments of suitableMPPs are provided in Table 1 and Example 5.

[0054] The MPP and/or compound of interest may be chemically synthesizedseparately, for example, by chemical synthetic routes and usingcommercially available reagents. Alternatively, if the MPP and/orcompound of interest is a polypeptide, it may be synthesized byrecombinant technology and purified according to known methods. Hostcells, cloning vectors, promoters and oligonucleotide linkers are wellknown and commercially available. Methodologies for using recombinanttechnology and purification methods are also well known, see CurrentProtocols in Molecular Biology, 4 Vols. Wiley. Generally, recombinanttechnology is preferred, as it is more amenable to larger scaleproduction and is more economical for mass production. Alternatively,MMPs may be obtained by specific protease degradation of a precursorproteins.

[0055] The compound of interest may be attached or linked to the MPP viachemical crosslinking at the N- or C-terminus of the MPP to create aconjugated (also referred to a a fusion) MPP and compound of interest,for example, via disulfide or ester linkages. In an alternativeembodiment, if the compound of interest is a peptide, the peptide may besynthesized by recombinant technology with a host cell with anexpression vector encoding a fusion of the MPP sequence and the compoundof interest under conditions to permit expression of the vector andobtaining the fusion MPP and compound of interest.

[0056] In another embodiment, the MPP and the compound of interest maybe attached or linked via a chemical linker. Chemical linkers are wellknown in the art, and include but are not limited todicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS),maleiimidobenzoyl-N-hydroxysuccinimide ester (MBS),N-ethyloxycarbonyl-2-ethyloxy-1,2-dihydroquinoline (EEDQ),N-isobutyloxy-carbonyl-2-isobutyloxy-1,2-dihydroquinoline (IIDQ).Preferred linkers may also be monomeric entities such as a single aminoacid, especially preferred are those amino acids with small side chains,or a small polypeptide chain, or polymeric entities of several aminoacids. Preferred polypeptide linkers are fifteen amino acids or less,more preferred are polypeptide linkers of ten or less amino acids. Evenmore preferred are polypeptide linkers of five or less amino acids. Inan alternative embodiment, the linker may be a nucleic acid encoding asmall polypeptide chain; preferred linkers encode a polypeptide offifteen or less amino acids. More preferred linkers are nucleic acidsencoding a small polypeptide chains of ten or less amino acids. Evenmore preferred linkers are nucleic acid encoding a small polypeptide offive or less amino acids, such as Gly-Phe-Leu-Gly, Gly-Gly, Gly-Leu orGly, and the like.

[0057] Recombinant technology may be used to express a fusion MPP,linker and compound of interest, as described above and is well known inthe art.

[0058] In another embodiment, the linker may be a cleavable linker,resulting in cleavage of the MPP and compound of interest once deliveredto the tissue or cell of choice. In such an embodiment, the cell ortissue would have endogenous (either naturally occurring enzyme or berecombinantly engineered to express the enzyme) or have exogenous (e.g.,by injection, absorption or the like) enzyme capable of cleaving thecleavable linker. Suitable enzymes for cleavage include, for example,use of a KEX2 protease recognition site (Lys, Arg) inserted betweenglucoamylase and the desired polypeptide to allow in vivo release of thedesired polypeptide from the fusion protein as a result of the action ofa native Aspergillus KEX2-like protease. (Contreras et al., 1991;Broekhuijsen et al., 1993; Ward et al., 1995). Another example of acleavable linker peptide comprises the recognition sequenceAsp-Asp-Asp-Asp-Lys, and wherein said fusion protein is cleavable byenterokinase.

[0059] Alternatively, the linker may be biodegradable such that thecompound of interest is detached from the fusion MPP and compound ofinterest by hydrolysis and/or enzymatic cleavage inside cells. Forexample, tumors often express specific proteases, and be used in thedelivery of prodrugs of cytotoxic agents. The linker may be selectivefor lysosomal proteases, such as cathepsin B, C, or D. Delivery ofprodrugs and their subsequent activation is well recognized, and such anapproach provides significantly less systemic toxicity due to prematurelinker hydrolysis in the blood, consequently a greater amount ofcompound of interest, i.e., drug or cytotoxic agent, is delivered to thetumor site. See for example, T. Higuchi and V. Stella provide a thoroughdiscussion of the prodrug concept in Pro-drugs as Novel DeliverySystems, Vol. 14 of the A.C.S. Symposium Series, American ChemicalSociety (1975). Examples of readily-cleavable groups include acetyl,trimethylacetyl, butanoyl, methyl succinoyl, t-butyl succinoyl,ethoxycarbonyl, methoxycarbonyl, benzoyl, 3-aminocyclohexylidenyl, andthe like.

[0060] The compound of interest is any organic molecule, and includessmall organic molecules, peptides, lipoproteins, and other modifiedproteins, polysaccharides, oligonucleotides, antisense oligonucleotides,and any other compound thought to have pharmaceutical, prophylactic,diagnostic properties and/or research interest. The compound of interestmay be a small organic molecule already known to have pharmaceuticalproperties, and thus the present invention may be used as a method oftreating a patient with the compound of interest. Alternatively, thecompound of interest may be a novel protein of unknown function, andthus the present invention may be used as a method of identifying thefunction of the compound of interest. In another embodiment, thecompound of interest may be an antisense molecule, and thus the presentinvention may be used as a method of altering transcription. In yetanother embodiment, the compound of interest may be a prodrug, e.g. inan inactive form but capable of being activated once within the cell. Inanother embodiment, the compound of interest may be a cytotoxic agent,and thus the invention may be used as a method of delivering a cytotoxicagent to a cell. The compound of interest also includes detectableproteins which are useful to generate conjugated MMP and the detectableprotein for identification of new MMPs. Detectable proteins include GFP,beta galactosidase, radiolabeled proteins and biotinylated proteins,proteins capable of conferring a detectable phenotype in the cell.

[0061] The present invention may be used to deliver the compound ofinterest into a cell in vitro, ex vivo or in vivo. For example, deliverymay be carried out in vitro by adding the conjugated MPP and compound ofinterest extracellularly to cultured cells. Delivery may be carried outex vivo by adding the conjugated MPP and compound of interestextracellularly or exogenously to a cultured sample removed from apatient, for example, blood, tissue or bone marrow, and returning thetreated sample to the patient. Delivery may be carried out in vivo byadministering the conjugated MPP and compound of interest by transdermaladministration, inhalation, or injection to a patient.

[0062] Any type of cell may used in the present invention. The cell maybe of mammalian, bacterial, viral or yeast origin. The cell may be acultured cell such as commonly used for oncology screening. Examples ofcultured cells include CHO, HEK293T, HeLa, and NIH3T3. The cell may be acultured cell from a patient suitable for ex vivo treatment with an MPPconjugate and reintroduction into a patient. The cell may be from thesame or different patient than the patient to be treated.

[0063] Compositions of the invention comprising the conjugated MPP andcompound of interest may be used for therapeutic, prophylactic,diagnostic or research purposes. Compositions may further compriseadjuvants, stabilizers and the like to improve the handling, stabilityand storage properties of the compositions.

[0064] Methods to identify novel MPPs are also part of the presentinvention. One method for identification of a membrane penetratingpeptide is to generate a conjugate peptide comprising the sequence—(X—X—X—X)_(n)— where n is an integer 1 to 7, and X each time isindependently selected from the group consisting of arginine, histidineor lysine, with a detectable protein such as GFP, beta galactosidase andthe like, adding the conjugate peptide to a cell and determining if theconjugated peptide is located within the cytoplasm and/or nucleus of thecell. Another method for identification of a membrane penetratingpeptide is to generate a conjugate peptide comprising a peptide derivedfrom or overlapping with a nuclear localization sequence of a mammalianor yeast protein and a detectable protein such as GFP, betagalactosidase and the like, adding the conjugate peptide to a cell anddetermining if the conjugated peptide is located within the cytoplasmand/or nucleus of the cell.

[0065] The following abbreviations are used for amino acids:

[0066] A refers to Ala, or alanine;

[0067] C refers to Cys or cysteine;

[0068] D refers to Asp or aspartic acid;

[0069] E refers to Glu or glutamic acid;

[0070] F refers to Phe or phenylalanine;

[0071] G refers to Gly or glycine;

[0072] H refers to His or histidine;

[0073] I refers to Ile or isoleucine;

[0074] K refers to Lys or lysine;

[0075] L refers to Leu or leucine;

[0076] M refers to Met or methionine;

[0077] N refers to Asn or asparagine;

[0078] P refers to Pro or proline;

[0079] Q refers to Gln or glutamine;

[0080] R refers to Arg or arginine;

[0081] S refers to Ser or serine;

[0082] T refers to Thr or threonine;

[0083] V refers to Val or valine;

[0084] W refers to Trp or tryptophan;

[0085] Y refers to Tyr or tyrosine.

[0086] Proteins are written with the N-terminus to the left.

[0087] The following abbreviations are used: ‘v/v’ refers to volume tovolume; ‘EYFP’ refers to a peptide fragment of the sequenceGlu-Tyr-Phe-Pro; ‘ORF’ refers to Open Reading Frame; ‘PCR’ refers topolymerase chain reaction; ‘CHO’ refers to Chinese Hamster Ovary cells;‘HEK293T’ refers to Human Embryonic Kidney cells, ‘HeLa’ refers toepithelial adenocarcinoma cells; ‘NIH3T3’ refers to Swiss mouse embryofibroblast cells; ‘DMSO’ refers to dimethyl sulfoxide; ‘FCS’ refers tofetal calf serum; ‘DMEM’ refers to Dulbecco's Modified Eagle's Medium;‘PBS’ refers to Phosphate buffered saline; ‘BSA’ refers to bovine serumalbumin; ‘C-terminus’ refers to the carboxy-terminus; ‘N-terminus’refers to the amino-terminus; ‘PTD’ refers to Peptide transductiondomain; ‘GPCR’ refers to G-protein coupled receptor; ‘TM’ refers to atransmembrane domain of a GPCR; ‘I’ refers to an intracellular loop of aGPCR; ‘5HT2A’ refers to serotonin receptor 2A; and ‘mAb’ refers tomonoclonal antibody.

EXAMPLES Example 1

[0088] Identification of an NLS within hPER1

[0089] Plasmid Construction

[0090] All hPer1 fragments described here are cloned as in-frameC-terminal fusion to EYFP. EYFP-hPer1 ORF, P1-N and P1-NX (FIG. 1A) isgenerated by insertion of EcoRI and XhoI digested fragments into EYFP-C1vector (Clontech). The other fragments are PCR amplified from thefull-length hPer1 cDNA and subcloned into EYFP-C1 vector. The first andthe last residue present in each of fragment is indicated in FIG. 1A.All constructs are verified by automated DNA sequencing.

[0091] Cell Culture and DNA Transfection

[0092] CHO, HeLa and 293T cells are maintained in Dulbecco's ModifiedEagle's Medium (DMEM) supplemented with 10% fetal calf serum (FCS), 50units/ml penicillin, 50 μg streptomycin, and 4 mM L-glutamine (hereafterreferred to as complete DMEM) at 37° C. with 5% CO₂. Transfection of thecells is carried in two-well Lab-Tek coverslips (Nunc Inc.) withLIPOFECT-AMINETM™ Reagent (Life Technologies) according to themanufacturer's instructions.

[0093] Peptides and Peptide Internalization

[0094] Peptides are synthesized by a commercial vendor (Bio Synthesis).For peptides internalization, cells are plated into two-well Lab-Tekcoverslips (Nunc Inc.) at a density of 2×10⁵ cells/well and culturedovernight. The peptides are dissolved in DMSO diluted to indicatedconcentration with PBS. The cell monolayers were incubated with theappropriate peptide/PBS solution at 1 μM standard concentration for 10min at room temperature (RT) unless otherwise specified. For experimentsat 4° C., the protocol was the same except that all incubations wereperformed at 4° C. until the end of the fixation procedure.

[0095] Immunofluorescence and Microscopy

[0096] For direct detection of expression and subcellular localizationof EYFP fusion protein, transfected cells were examined directly withoutfixation or after fixation with 4% (v/v) formaldehyde in PBS for 20 minat 4° C. and washed with PBS. For indirect immunodetection ofbiotinylated peptides, fixed cell were washed twice with PBS andpermeabilized with 0.3% Triton X-100 in PBS for 20 min at 4° C. andblocked with 2% BSA in PBS for 30 min at RT. Cells were then washed withPBS and incubated with Streptavidin-FITC™ (Sigma) or -Alex499 (MolecularProbe), 1:400 diluted in 0.2% Tween 20, 2% BSA in PBS for 1 h at RT.Following 2×5 min washes with PBS and once with 0.3% Triton X-100 in PBSfor 20 min RT. In some experiment, the nucleus was stained with 50 ng/mlHoechst 33258 (Sigma) or 3 μg/ml propidium iodide in PBS. Thesubcellular localization of the fluorescence was analyzed on an Olympusmicroscope. Confocal images were taken on a Zeiss confocal laser scanmicroscope (CLSM phoibos 1000).

[0097] Though it is known that nuclear entry of PER1 is important forits function, no putative NLS was identified using a standard ProfileScanning program (Shearman, L. P., et al., Neuron 19, 1261-1269 (1997),Yagita, K., et al., Genes Dev. 14, 1353-1363 (2000)). To determine theNLS of hPER1 experimentally, three full-length hPER1 (P1-FL) wereconstructed and denoted as P1-N, P1-NM and P1-C (FIG. 1A). The abilityof these constructs to localize to the nucleus in CHO cells thenanalyzed. An EYFP-tag was used to facilitate the detection of hPER1 inliving cells; however, the EYEP-tag had no apparent contribution onhPER1 fusion protein localization since hPER1 constructs made with anN-terminal Flag-tag presented an identical cytological distributionpattern (data not shown). After transient transfection, both P1-FL andP1-NM proteins were expressed in the nucleus of transfected cells asearly as 10 hours post-transfection, while both P1-N and P1-Caccumulated only in the cytoplasm (FIG. 1B). The EYFP vector control wasdiffuse in both the nucleus and cytoplasm. These results demonstratethat a functional NLS in hPER1 is located between P1-N and P1-C in whatwe designated as region M (see FIG. 1A).

[0098] To farther localize the NLS in region M (amino acids 481-890), aseries of 8 deletion constructs, P1-F1 to P1-F8, were generated and thesubcellular distribution of each mutant was assayed as indicated inFIGS. 1A and B. Sequential deletion from amino acid 581 (P1-F2) toposition 821(P1-F7) of region M resulted in nuclear localization.Further deletion of amino acids 821 to 841 (P1-F8) resulted in adiffused fluorescent pattern within transfected cells with alocalization pattern similar to that of the EYFP vector control. Thesedata indicate that a NLS exists between amino acids 821 and 890, and islocated at the C-terminus of region M. This observation was confirmed bythe construction of an additional EYFP fusion protein, P1-NLS, whichcontained hPER1 amino acids 830-845. This region contains a string ofbasic residues that might function as a NLS (Weis, K., Trends Biochem.Sci. 23, 185-189 (1998), Truant, R. and Cullen, B. R. Mol Cell Biol. 19,1210-1217 (1999)). As expected, P1-NLS exhibited nuclear localization in100% of transfected cells (FIG. 1B). Other regions of PER1 in additionalfusion constructs failed to localize to the nucleus (data not shown).Therefore, we conclude that the NLS of hPER1 (hPER1-NLS) is localized towithin amino acids 830-845. Interestingly, construct P1-F1 has astrictly cytoplasmic localization pattern irrespective of the fact thatit contains the NLS, supporting published observations that this regionalso contains and as yet unidentified cytoplasmic localization domain(Vielhaver, E., et al., Mol Cell Biol. 20, 4888-4899 (2000)). Sequencealignment shows that the hPER1-NLS is conserved between human and mousePER1 proteins (FIG. 1A), but not with other putative NLSs, or with otherhuman, mouse or Drosophila PERs. After completion of our studies,Vielhaver et al. (2000), identified a longer mouse PER1-NLS thatcontains our identified 16 amino acid sequence (Vielhaver, E., et al.,Mol Cell Biol. 20, 4888-4899 (2000)); thus, supporting our findings.

Example 2

[0099] hPER1-NLS Encodes an MPP

[0100] Two common features of the three identified gene encoded MPPs(TAT, Antp, and VP22) are that they are derived from nuclear proteinsand they consist of basic amino acid residues (Lindgren, M., et al.,Trends Pharmacol Sci. 3, 99-103 (2000)). hPER1 is also a nuclear proteinwhose NLS is rich in basic amino acids (SRRHHCRSKAKRSRHH, see FIG. 1).These similarities led us to determine whether hPER-NLS might alsofunction as a MPP. In order to test this hypothesis, we synthesizedseveral N-terminally biotinylated peptides: hPER1-MPP, Flag-taggedhPER1-MPP, Flag-tagged TAT-PTD, Flag-Flag alone, See Table 1 below:TABLE 1 Nuclear Localizati Transducing on Fusion Name Amino AcidSequence Peptide ¹ Protein ² hPER1 GRRHHCRSKAKRSRHH + + Flag-hPER1GMDYKDDDDKGSRRHHCRSK + nd AKRSHH Flag-TAT GMDYKDDDDKGYGRKKKRR + + QRRRFlag GMDYKDDDDKG − − MDYKDDDDK Antennapedia GRQIKIWFQNRRMKWKK + nd 9Arginine GRRRRRRRRR + nd 9 Lysine GKKKKKKKKK + nd 9 Histidine GHHHHHHHHH− nd NLSs: SV40 GDPKKKRKV − + hPER2 GKKTGKNRKLKSKRVKPRD − + hPER3GRKIGKHKRKKLP + + Thyroid A-1 GKRVAKRKLIEQNRERRR + + HME-1GRKLKKKKNEKEDKRPRT + + ABL-1 GKKTNLFSALIKKKKTA + + Nucleoplasmin XGRRERNKMAAAKCRNRRR + + C-FOS GRRERNKMAAAKCRNRRR − + GCN-4GKRARNTEAARRSRARKL + + [R/H/K]- [R/H/K]- [R/H/K]- [R/H/K] HEN1/NSLC1GRRRRATAKYRTAH + + HEN2/NSLC2 GKRRRRATAKYRSAH + + HNF3 GRRRRKRLSHRT + +cAMP dependent GRRRRRERNK + + TF Cyclin L ania-6a GKHRHERGHHRDRRER − +beta Zip TF GKKKRKLSKRESAKRSR − + GFP nd −

[0101] Fn 1: Results shown for selected MPPs, see FIG. 5

[0102] Fn 2: Results shown for selected MPPs, see FIG. 5

[0103] The peptides are assayed for their ability to penetrate cellularmembranes. Intracellular localization is assayed by direct staining withlabeled Streptavidin ALEXA reagents or by indirect staining withanti-Flag mAb followed by the addition of labeled secondary antibodies.When added to the cells in culture at a concentration of 10 μM,hPER1-MPP, Flag-hPER1-MPP and Flag TAT-PTD peptides are found topenetrate rapidly into 100% cells (FIG. 2A and FIG. 5). By bothdetection methods, hPER1-MPP, Flag-tagged hPER1-MPP, and Flag-taggedTAT-PTD are observed to be diffusely distributed throughout thecytoplasm, but concentrated within subnuclear domains that appear asdistinct foci within the nucleoplasm and the nucleolus. In contrast,biotinylated negative control peptides, Flag-Flag and several additionalpeptides derived from other hPER1 regions are only barely discerniblebackground staining, with no staining in the nucleus or nucleoli, evenat high concentrations (data not shown). Confocal microscopy is used toconfirm the intracellular and intranuclear staining of Flag-taggedhPER1-MPP, and that the negative control peptides are not internalized(FIG. 2A).

[0104] hPER1-MPP rapidly penetrated the cellular membranes and localizedin nuclear regions with efficiencies similar to the TAT-PTD peptide(FIG. 2B). Identical results are obtained using CHO, HEK293T, HeLa,NIH3T3 and cultured rat primary cortical neurons (data not shown),indicating cell type-independent penetration.

[0105] hPER1-MPP internalization occurs rapidly (within 5 min), withsimilar potencies at 4C. and 37C. and even after cell membrane fixation(data not shown). Thus, the amino acid sequence 830-845 of hPER1functions as both as a protein nuclear/nucleolar localization signal inthe fusion protein and as a MPP, and that membrane penetration isindependent of traditional receptor-mediated endocytic mechanisms.

Example 3

[0106] Arginine 7 is Essential for hPER1-MPP Activity

[0107] To date, the mechanisms as well as the structural basis wherebyMPPs transverse cellular membranes have not been elucidated. Wetherefore sought to determine if there were key residues withinhPER1-MPP that were important for maintaining those properties essentialfor its membrane penetrating potential. We separately replaced eachamino acid in hPER1-MPP to alanine (Table 2), and assayed for theability of these mutated peptides to penetrate living cells relative tothe wild-type hPER1-MPP.

[0108] Alanine scanning: Transducing Name hPER1 -PTD alaninesubstitution Peptide hPER1-PTD S R R H H C R S K A K R S R H H + R2A S AR H H C R S K A K R S R H H + R3A S R A H H C R S K A K R S R H H + H4AS R R A H C R S K A K R S R H H + H5A S R R H A C R S K A K R S R H H +C6A S R R H H A R S K A K R S R H H + R7A S R R H H C A S K A K R S R HH − S8A S R R H H C R A K A K R S R H H + K9A S R R H H C R S A A K R SR H H + K11A S R R H H C R S K A A R S R H H + R12A S R R H H C R S K AK A S R H H + S13A S R R H H C R S K A K R A R H H + R14A S R R H H C RS K A K R S A H H + hPER1- R R H H C R S K A K R S R + PTD13 hPER1-QELSEQIHRLLLQPV − Control (484- 503)

[0109] As shown in FIG. 3, most of the single alanine substitutions hadvery little effect on membrane penetrating capabilities as compared withwild-type peptide. However, changing arginine 7 to an alanine (R7A)reduced the detectable cytological signal to that observed for thenegative control peptides. Thus, the arginine 7 to alanine mutationsignificantly reduced the membrane penetrating properties of hPER1-MPP.Identical observations were observed after changing arginine 7 toglutamic acid (R7E) (data not shown). Furthermore, the simultaneousdeletion of the N-terminal serine and of the two C-terminal histidinesfrom hPER1-MPP (hPER1-MPP 13) had little overall effect on the positivemembrane penetrating potential of the peptide (FIG. 3).

[0110] The arginine 7 residue plays a critical role in the cellpenetrating ability of the hPER1-MPP. We therefore sought to determineif the R7A mutation affected nuclear translocation of a fusion proteinP1-NLS. CHO cells transfected with fusion protein P1-R7A (arginine 7mutated to alanine) have intense nuclear staining similar to thewild-type, P1-NLS (data not shown). Nuclear translocation appears to benormal in the P1-R7A mutant fusion protein, but subnuclear targeting tothe nucleoli is disrupted (data not shown). These data indicate thatmembrane penetration and nucleoli targeting are affected by the singleR7 amino acid residue and indicate that nuclear translocation ofhPER1-NLS has separate and distinct determinants.

Example 4

[0111] hPER1-MPP Delivery of Functioning Molecules

[0112] One of the features of MPPs is their ability to cargo proteinsand peptides into cells. We were successful in coupling hPER1-MPP toB-galactosidase and in delivering the fusion protein into cells inculture (data not shown), as has been described by Fawells et al., 1994(Fawell, S., et al., Proc Natl Acad Sci USA. 91, 664-668 (1994)).However, to further extend the functional utility of MPPs, we testedhPER1-MPP in fusion with a physiologically relevant and biologicallyactive peptide. Wess and colleagues (1993) have shown a functional rolefor the conserved transmembrane segment 7 (TM7) of the G-protein coupledreceptor (GPCR) superfamily. Along with TM7, the third intracellularloop (I3) plays a significant role in GPCR calcium signaling (Wess, JM., et al., EMBO J. 12, 331-338 (1993)) while intracellular loops 1 and2 (I1 and I2) appear not to be important. Using the serotonin receptor,5HT2A, we experimentally tested the ability of hPER1-MPP and TAT-PTD infusion with peptides designed from I1 and the TM7 domains to activatethe receptor. Biotinylated peptides hPER1-MPP TM7, TAT-PTD TM7,hPER1-MPP I1, TAT-PTD I1, hPER-MPP, TAT-PTD, TM7 or I1 were incubated ata concentration of 10 μM with a 5HT2A receptor CHO stable cell line.Peptide membrane penetration was assayed using Streptavidin-Alexa 594 asdescribed above. As shown in FIG. 4A, receptor signaling is activated bythe addition of exogenous serotonin, hPER1-MPP TM7, and TAT-PTD TM7 asmeasured by the level of the calcium response. However, TM7 alone norany of the other peptides were able to generate a calcium response.Furthermore, the activation of the receptor by hPER1-MPP TM7 and TAT-PTDTM7 is peptide concentration dependent, FIG. 4B. The addition ofincreasing concentrations of the activating peptide, TM7, in fusion withhPER1-MPP or TAT-PTD results in a calcium response in a dose dependentmanner. TAT-PTD TM7 appears to be a more potent 5HT2A receptor activatorthan is hPER1-MPP TM7. A simple explanation for this result is thatTAT-PTD TM7 is more cytoplasmically localized or has greater cellpenetrating capabilities than hPER1-MPP TM7, although we have notobserved that to be the case. Similar results were also obtained in thislaboratory using hPER1-MPP in fusion with β-adrenergic activatingpeptides (unpublished data). These data support previous results thathPER1-MPP not only penetrates cell membranes, but also demonstrates thatit is capable of cargoing peptides to intracellular compartments toinitiate biologically relevant signal transduction events.

Example 5

[0113] Identification of Other Gene Encoded MPPs

[0114] Since hPER1 is a nuclear protein proposed to be involved intranscriptional regulation, and since, to date, all PTDs derived fromnaturally occurring proteins are transcription factors (TAT, Antp, andVP22), we sought to determine if other PTD sequences existed within thegenome. To this end, we used two approaches; first, we searched the NCBInon-redundant protein database for all known and putative NLS's (table1, 10-17). We synthesized peptides corresponding to the NLS amino acidsequences and assayed for peptide transduction. As shown in table 1 andFIG. 5, 6 of the 7 peptides synthesized had membrane penetratingcharacteristics similar to hPER-PTD and TAT-PTD. These proteins includedhuman proteins of the thyroid hormone receptor alpha-1, homeobox proteinHME1, and proto-oncogene protein ABL-1. Furthermore, (table 1 and FIG.5) when we create in frame fusion proteins between these peptidesequences and GFP then transfected into CHO or HEK 293T cells, all ofthe sequences conferred nuclear localization of the fusion protein.

[0115] Our second approach to identifying PTDs involved searching theNCBI non-redundant protein database collection with a degenerativealgorithm (see FIG. 5, legend). Using these search parameters, we found533,291 sequences of which the conditions for the algorithm weresatisfied 129,169 times (24%). By limiting our search to include either“transcription factors, cytokines or tyrosine kinases”, we identified8280 transcription factor protein sequences of which the algorithmpattern occurred 7374 times (89%); within 2333 cytokine proteinsequences the pattern occurred 450 times (19%); and within 2513 tyrosinekinase protein sequences the pattern occurred 843 times (36%). Becausethe algorithm occurred most frequently in nuclear proteins, wesynthesized peptides to putative PTDs for 6 of the “transcriptionfactor” sequences and assayed for their ability to penetrate into thecells. As shown in table 1, results in lines 18-23 and FIG. 3A, 4 of the6 peptides tested had membrane penetrating properties similar tohPER1-PTD and TAT-PTD. These proteins included two human proteinsHEN1/NSLC-1 and HEN2/NSLC-2 which are reported to be involved inneuronal differentiation and development (Uittenbogaard, M., Peavy, D.R. and Chiaramello, A. 1999. Expression of the bHLH gene NSCL-1 suggestsa role in regulation of cerebellar granule cell growth anddifferentiation. J. Neurosci. Res. 57:770-781, Lipkowitz, S. et al.1992. A comparative structural characterization of the human NSCL-1 andNSCL-2 genes. Two basic helix-loop-helix genes expressed in thedeveloping nervous system. J. Biol. Chem. 267:21065-21071), rat HNF-3(17), and a Drosophila cAMP dependent transcription factor (18).Furthermore, (table 1 and FIG. 5) when we create in frame fusionproteins between these peptides and GFP and transfected into CHO or HEK293T cells, all of the sequences conferred nuclear localization of thefusion protein. These results indicate that PTD sequences can be foundwithin or overlapping with NLSs. However not all NLSs are PTDs as isapparent in SV40, hPER2, C-FOS, Cyclin L ania-6 and beta Ziptranscription factor NLSs (table 1). These results also suggest thatPTDs sequences are prevalent throughout the genome and in particularwithin nuclear proteins.

Example 6

[0116] hPER-PTD with β-Galactosidase

[0117] At least one feature of HIV TAT transducing peptide is itsability to cargo proteins into cells and tissues. We therefore sought todetermine if hPER1 transducing peptide could cargo beta galactosidaseinto cells. To perform this experiment, we followed a protocol byFrankel et al. PNAS 1989 (19):7397-401, whereby, we chemically linkedhPER1-PTD or hPER-PTD R7A(with Ala replacing Arg⁷) to full lengthβ-galactosidase and assayed for the ability of these conjugates andbeta-galactosidase protein alone to transduce into CHO cells. As shownin the FIG. 6, panel 1, cells incubated with hPER-PTD β-galactosidasefusion showed positive enzymatic activity for β-galactosidase asindicated by the blue color in the cells after the addition of X-gal.However, neither hPER-MPP R7A β-galactosidase nor β-galactosidaseprotein alone was able to enter the cells as indicated by a no bluestaining reactivity after the addition of X-gal, panels 2 and 3. Thesedata indicate that like TAT peptide, hPER1-PTD can cargo a large (120kD) protein into cells.

1 54 1 10 PRT Artificial Sequence Sequence of nuclear location sequencecontained within the N-terminal of IL-alpha propiece. 1 Asn Gly Lys ValLeu Lys Lys Arg Arg Leu 1 5 10 2 16 PRT Artificial Sequence Signalsequence peptide from Antennapedia homeodomain 2 Arg Gln Ile Lys Ile TrpPhe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 3 15 PRT ArtificialSequence Fibroblast growth factor signal sequence peptide 3 Ala Ala ValAla Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala 1 5 10 15 4 29 PRTArtificial Sequence HIV tat signal sequence peptide 4 Cys Phe Ile ThrLys Ala Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg 1 5 10 15 Arg Gln ArgArg Arg Pro Pro Gln Gly Ser Gln Thr His 20 25 5 4 PRT ArtificialSequence Peptide sequence of an N-terminal fluorescein isothiocyanate(FITC) peptide motif 5 Gly Gly Gly Gly 1 6 7 PRT Artificial SequenceFragment of IFN-gamma 6 Arg Lys Arg Lys Arg Ser Arg 1 5 7 7 PRTArtificial Sequence Fragment of the N-terminus of fibroblast growthfactor. 7 Asn Tyr Lys Lys Pro Lys Leu 1 5 8 8 PRT Artificial SequenceLuinus luteus nuclear protein import sequence 8 Lys Pro Lys Lys Lys LysGlu Lys 1 5 9 5 PRT Artificial Sequence Sequence of the basic motif inthe nuclear protein import sequence of Smad 3 protein 9 Lys Lys Leu LysLys 1 5 10 11 PRT Artificial Sequence Sequence of intracellular loop of5HT2A receptor 10 Ser Leu Glu Lys Lys Leu Gln Asn Ala Thr Asn 1 5 10 1123 PRT Artificial Sequence Sequence of C-terminal transmembrane 7 domainderived from 5HT2A receptor 11 Lys Thr Tyr Arg Ser Ala Phe Ser Arg TyrIle Gln Tyr Lys Glu Asn 1 5 10 15 Lys Lys Pro Leu Gln Leu Ile 20 12 9PRT Artificial Sequence Fragment of HIV TAT 12 Arg Lys Lys Arg Arg GlnArg Arg Arg 1 5 13 4 PRT Artificial Sequence Synthetic peptide 13 GlyPhe Leu Gly 1 14 5 PRT Artificial Sequence Synthetic peptide 14 Asp AspAsp Asp Lys 1 5 15 4 PRT Artificial Sequence synthetic peptide 15 GluTyr Phe Pro 1 16 16 PRT Artificial Sequence Nuclear protein importsequence of hPER1 16 Ser Arg Arg His His Cys Arg Ser Lys Ala Lys Arg SerArg His His 1 5 10 15 17 16 PRT Artificial Sequence Synthetic Peptide 17Gly Arg Arg His His Cys Arg Ser Lys Ala Lys Arg Ser Arg His His 1 5 1015 18 23 PRT Artificial Sequence Synthetic peptide 18 Gly Met Asp TyrLys Asp Asp Asp Asp Lys Gly Tyr Gly Arg Lys Lys 1 5 10 15 Lys Arg ArgGln Arg Arg Arg 20 19 23 PRT Artificial Sequence Synthetic peptide 19Gly Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Tyr Gly Arg Lys Lys 1 5 1015 Lys Arg Arg Gln Arg Arg Arg 20 20 19 PRT Artificial SequenceSynthetic peptide 20 Gly Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Met AspTyr Asp Asp 1 5 10 15 Asp Asp Lys 21 17 PRT Artificial SequenceSynthetic peptide 21 Gly Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg MetLys Trp Lys 1 5 10 15 Lys 22 10 PRT Artificial Sequence Syntheticpeptide 22 Gly Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 10 23 10 PRTArtificial Sequence Synthetic peptide 23 Gly Lys Lys Lys Lys Lys Lys LysLys Lys 1 5 10 24 10 PRT Artificial Sequence Synthetic peptide 24 GlyHis His His His His His His His His 1 5 10 25 9 PRT Artificial SequenceSynthetic peptide 25 Gly Asp Pro Lys Lys Lys Arg Lys Val 1 5 26 19 PRTArtificial Sequence Synthetic peptide 26 Gly Lys Lys Thr Gly Lys Asn ArgLys Leu Lys Ser Lys Arg Val Lys 1 5 10 15 Pro Arg Asp 27 12 PRTArtificial Sequence Synthetic peptide 27 Gly Arg Lys Gly Lys His Lys ArgLys Lys Leu Pro 1 5 10 28 18 PRT Artificial Sequence Synthetic peptide28 Gly Lys Arg Val Ala Lys Arg Lys Leu Ile Glu Gln Asn Arg Glu Arg 1 510 15 Arg Arg 29 18 PRT Artificial Sequence Synthetic peptide 29 Gly ArgLys Leu Lys Lys Lys Lys Asn Glu Lys Glu Asp Lys Arg Pro 1 5 10 15 ArgThr 30 17 PRT Artificial Sequence synthetic peptide 30 Gly Lys Lys ThrAsn Leu Phe Ser Ala Leu Ile Lys Lys Lys Lys Thr 1 5 10 15 Ala 31 18 PRTArtificial Sequence Synthetic peptide 31 Gly Arg Arg Glu Arg Asn Lys MetAla Ala Ala Lys Cys Arg Asn Arg 1 5 10 15 Arg Arg 32 18 PRT ArtificialSequence Synthetic peptide 32 Gly Lys Arg Ala Arg Asn Thr Glu Ala AlaArg Arg Ser Arg Ala Arg 1 5 10 15 Lys Leu 33 14 PRT Artificial SequenceSynthetic peptide 33 Gly Arg Arg Arg Arg Ala Thr Ala Lys Tyr Arg Thr AlaHis 1 5 10 34 15 PRT Artificial Sequence Synthetic peptide 34 Gly LysArg Arg Arg Arg Ala Thr Ala Lys Tyr Arg Ser Ala His 1 5 10 15 35 12 PRTArtificial Sequence Synthetic peptide 35 Gly Arg Arg Arg Arg Lys Arg LeuSer His Arg Thr 1 5 10 36 10 PRT Artificial Sequence Synthetic peptide36 Gly Arg Arg Arg Arg Arg Glu Arg Asn Lys 1 5 10 37 16 PRT ArtificialSequence Synthetic peptide 37 Gly Lys His Arg His Glu Arg Gly His HisArg Asp Arg Arg Glu Arg 1 5 10 15 38 17 PRT Artificial SequenceSynthetic peptide 38 Gly Lys Lys Lys Arg Lys Leu Ser Asn Arg Glu Ser AlaLys Arg Ser 1 5 10 15 Arg 39 16 PRT Artificial Sequence Syntheticpeptide 39 Ser Arg Arg His His Cys Arg Ser Lys Ala Lys Arg Ser Arg HisHis 1 5 10 15 40 16 PRT Artificial Sequence Synthetic peptide 40 Ser AlaArg His His Cys Arg Ser Lys Ala Lys Arg Ser Arg His His 1 5 10 15 41 16PRT Artificial Sequence Synthetic peptide 41 Ser Arg Ala His His Cys ArgSer Lys Ala Lys Arg Ser Arg His His 1 5 10 15 42 16 PRT ArtificialSequence Synthetic peptide 42 Ser Arg Arg Ala His Cys Arg Ser Lys AlaLys Arg Ser Arg His His 1 5 10 15 43 16 PRT Artificial SequenceSynthetic peptide 43 Ser Arg Arg His Ala Cys Arg Ser Lys Ala Lys Arg SerArg His His 1 5 10 15 44 16 PRT Artificial Sequence Synthetic peptide 44Ser Arg Arg His His Ala Arg Ser Lys Ala Lys Arg Ser Arg His His 1 5 1015 45 16 PRT Artificial Sequence Synthetic peptide 45 Ser Arg Arg HisHis Cys Ala Ser Lys Ala Lys Arg Ser Arg His His 1 5 10 15 46 16 PRTArtificial Sequence Synthetic peptide 46 Ser Arg Arg His His Cys Arg AlaLys Ala Lys Arg Ser Arg His His 1 5 10 15 47 16 PRT Artificial SequenceSynthetic peptide 47 Ser Arg Arg His His Cys Arg Ser Ala Ala Lys Arg SerArg His His 1 5 10 15 48 16 PRT Artificial Sequence Synthetic peptide 48Ser Arg Arg His His Cys Arg Ser Lys Ala Ala Arg Ser Arg His His 1 5 1015 49 16 PRT Artificial Sequence Synthetic peptide 49 Ser Arg Arg HisHis Cys Arg Ser Lys Ala Lys Ala Ser Arg His His 1 5 10 15 50 16 PRTArtificial Sequence Synthetic peptide 50 Ser Arg Arg His His Cys Arg SerLys Ala Lys Arg Ala Arg His His 1 5 10 15 51 16 PRT Artificial SequenceSynthetic peptide 51 Ser Arg Arg His His Cys Arg Ser Lys Ala Lys Arg SerAla His His 1 5 10 15 52 13 PRT Artificial Sequence Synthetic peptide 52Arg Arg His His Cys Arg Ser Lys Ala Lys Arg Ser Arg 1 5 10 53 15 PRTArtificial Sequence Synthetic peptide 53 Gln Glu Leu Ser Glu Gln Ile HisArg Leu Leu Leu Gln Pro Val 1 5 10 15 54 4 PRT Artificial SequenceSynthetic peptide 54 Xaa Xaa Xaa Xaa 1

We claim:
 1. A fusion protein for delivery of a compound of interestinto a cell, comprising a membrane penetrating peptide attached to acompound of interest.
 2. The fusion protein according to claim 1,wherein the membrane penetrating peptide is derived from a nuclearlocalization sequence, overlaps with a nuclear localization sequence ofa mammalian or yeast protein or comprises a sequence —(X—X—X—X)_(n)—where n is an integer 1 to 7, and X each time is independently selectedfrom the group consisting of arginine, histidine or lysine.
 3. Thefusion protein according to claim 2, wherein the nuclear localizationsequence is derived from a nuclear protein or transcription factor. 4.The fusion protein according to claim 3, wherein the transcriptionfactor is a Period protein.
 5. The fusion protein according to claim 4,wherein the Period protein is a human Period protein.
 6. The fusionprotein according to claim 5, wherein the mammalian Period protein ishuman Period1 protein.
 7. The fusion protein according to claim 2wherein the membrane penetrating peptide comprises the sequence—(X—X—X—X)_(n)— where n is an integer 1 to 7, and X each time isindependently selected from the group consisting of arginine, histidineor lysine.
 8. The fusion protein according to claim 7, wherein n is aninteger 1 to
 4. 9. The fusion protein according to claim 8, wherein n isan integer 1 to
 2. 10. The fusion protein according to claim 1, whereinthe compound of interest is a peptide, protein, chemical entity, nucleicacid, or any modified form thereof.
 11. A method of delivering acompound of interest into a cell, comprising contacting a cell with afusion protein according to claim
 1. 12. The method of delivering acompound of interest into a cell in vitro, comprising contacting acultured cell with a fusion protein according to claim
 1. 13. The methodof delivering a compound of interest into a cell ex vivo, comprisingcontacting a cell with a fusion protein according to claim 1 andintroducing the cell into the body of a patient.
 14. The method ofdelivering a compound of interest into a cell in vivo, comprisingadministering to a patient a fusion protein according to claim
 1. 15. Amethod for identifying a membrane penetrating peptide, wherein a peptidecomprises a sequence —(X—X—X—X)_(n)— where n is an integer 1 to 7, and Xeach time is independently selected from the group consisting ofarginine, histidine or lysine, by generating a conjugate peptidecomprising the sequence —(X—X—X—X)_(n)— where n is an integer 1 to 7,and X each time is independently selected from the group consisting ofarginine, histidine or lysine, with a detectable protein, adding theconjugate peptide exogenously to a cell and determining if theconjugated peptide is located within the cytoplasm and/or nucleus of thecell.
 16. A method for identifying a membrane penetrating peptide,wherein a peptide comprises a sequence derived from or overlapping witha nuclear localization sequence of a mammalian or yeast protein, bygenerating a conjugate peptide comprising the part or all of the nuclearlocalization sequence with a detectable protein, adding the conjugatepeptide exogenously to a cell and determining if the conjugated peptideis located within the cytoplasm and/or nucleus of the cell.
 17. Themethod of delivering a compound of interest into a cell, comprisingadministering to a cell a fusion protein according to claim 1, whereinthe membrane penetrating peptide comprises a sequence —(X—X—X—X)_(n)—where n is an integer 1 to 7, and X each time is independently selectedfrom the group consisting of arginine, histidine or lysine.
 18. A fusionprotein for delivering a compound of interest into a cell, wherein thefusion protein comprises a membrane penetrating peptide comprising asequence —(X—X—X—X)_(n)— where n is an integer 1 to 7, and X each timeis independently selected from the group consisting of arginine,histidine or lysine, and a compound of interest.
 19. The fusion proteinof claim 18, wherein the compound of interest is directly chemicallyattached to the membrane penetrating peptide or by a linker.
 20. Thefusion protein of claim 19, wherein the linker is an amino acid linkeror a polypeptide linker.
 21. The fusion protein of claim 18, wherein themembrane penetrating protein is produced by recombinant technology,chemical synthesis or degradation of a precursor protein.